Helicases are molecular motor proteins that use energy from hydrolysis of ATP to manipulate DNA and RNA in all phases of nucleic acid metabolism. Numerous mutations have been identified in many different helicases that are associated with human diseases including cancer, heart disease, and neurological disorders. The Pif1 family of helicases has been identified in all eukaryotes and is involved in maintenance of both nuclear and mitochondrial genomes. Mutation in Pif1 can increase the risk for some forms of breast cancer. The primary function of helicases is to unwind duplex DNA, but other critical functions have been discovered for which biochemical mechanisms are unknown. Helicases can displace proteins from DNA in reactions that are critical for maintaining genomic stability. One of the overall goals of this proposal is to determine the mechanism(s) by which helicases push other proteins from DNA. Helicases not only unwind duplex DNA, but also unfold other forms of DNA such as G-quadruplex DNA (G4DNA). G4DNA is made up of guanine-rich sequences that have been implicated in gene expression, DNA replication, telomere maintenance, and mitochondrial DNA metabolism. Another goal of this proposal is to characterize the mechanism of G4DNA unfolding. We will use the prokaryotic helicase Dda and its eukaryotic homolog, Pif1 to determine the mechanisms for helicase- catalyzed protein displacement and unfolding of G4DNA. Based on preliminary data, we propose that mechanisms for Pif1-catalyzed unfolding of G4DNA and protein displacement are tightly regulated by cooperative binding to DNA. Within this overarching framework, more detailed hypotheses will be tested. In Aim 1, we will examine the role of a putative protein displacement loop in removing proteins from DNA. We will test the hypothesis that a stepwise mechanism is used to push proteins from DNA. We will determine the importance of cooperative binding of Pif1 to DNA during removal of proteins from telomeric DNA. In aim 2, we will determine the mechanism for unfolding of G4DNA using a combination of chemical perturbation along with single molecule Frster energy transfer (smFRET). We will use mutagenesis to examine the Pif1 signature sequence for binding to G4DNA. Using a proteomics screen, we discovered that Dbp2 helicase binds tightly to G4DNA. We will investigate Dbp2-catalyzed unfolding of G4RNA and G4 RNA-DNA hybrid molecules. In Aim 3, we will determine how single-stranded binding proteins Rim1 and RPA regulate the G4DNA unfolding activity of Pif1. We will determine whether Pif1 stimulates the polymerase activity of mitochondrial polymerase Mip1. We have developed a CRISPR-based method to isolate any desired gene segment and analyze that DNA for protein complexes. We will target chromatin that contains G4DNA sequences to identify proteins and chromatin markers that regulate these structures. This work will establish the mechanisms used by helicases to unfold G4DNA, displace proteins from DNA, and thereby regulate genomic stability.